HKI-272

Quality Control

Biological Activity

Neratinib is a potent irreversible inhibitor of epidermal growth factor receptor and human epidermal receptor 2 with IC50 of 92 nM and 59 nM, respectively. Neratinib inhibits the proliferation of EGFR-dependent A431 cells with an IC50 of 81 nM. Neratinib (HKI-272) also weakly inhibits tyrosine kinases KDR and Src with IC50 of 0.8 μM and 1.4 μM, respectively. Likewise, Neratinib inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin in a concentration-dependent manner (IC50 = 0.24 μM). Neratinib enhanced the sensitivity of ABCB1-overexpressing cells to ABCB1 substrates. It is noteworthy that Neratinib augmented the effect of chemotherapeutic agents in inhibiting the growth of ABCB1-overexpressing primary leukemia blasts and KBv200 cell xenografts in nude mice. Moreover, Neratinib increased doxorubicin accumulation in ABCB1-overexpressing cell lines and Rhodamine 123 accumulation in ABCB1-overexpressing cell lines and primary leukemia blasts. Neratinib (HKI-272) stimulated the ATPase activity of ABCB1 at low concentrations but inhibited it at high concentrations.

Protocol

Cell Experiment

Cell lines

3T3, 3T3/neu, A431, SK-Br-3, BT474, MDA-MB-435, and SW62 cells

Preparation method

Cell Proliferation Assays.Cells were plated in 96-well tissue culture plates (3T3, 3T3/neu, 5000 cells/well; A431, SK-Br-3, BT474, MDA-MB-435, and SW620, 10,000 cells/well). The following day, dilutions of compound (0.5 ng/ml–5 μg/ml) were added, and cells were cultured for 2 days (6 days for BT474). Cell proliferation was determined using sulforhodamine B, a protein binding dye. Briefly, cells were fixed with 10% trichloroacetic acid and washed extensively with water. Cells were then stained with 0.1% sulforhodamine B (Sigma-Aldrich) and washed in 5% acetic acid. Protein-associated dye was solubilized in 10 mm Tris, and absorbance was measured at 450 nm (Victor2). Inhibition of cell proliferation was calculated using the formula: percentage of inhibition = 100 − 100 (Td − To/Tc − To), where Td is the absorbance of drug treated cells, Tc is the absorbance of untreated cells, and To is the absorbance at the time of drug addition. To values were determined by plating cells separately and fixing them at the time of drug addition. The concentration of compound which inhibits cell proliferation by 50% (IC50) was determined from inhibition curves.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species

Mouse

Rat

Rabbit

Guinea pig

Hamster

Dog

Weight (kg)

0.02

0.15

1.8

0.4

0.08

10

Body Surface Area (m2)

0.007

0.025

0.15

0.05

0.02

0.5

Km factor

3

6

12

8

5

20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by

Animal B Km

Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the Km factor for a mouse and then divide by the Km factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Chemical Information

Molecular Weight

557.04

Formula

C30H29ClN6O3

CAS Number

698387-09-6

Purity

100.00%

Solubility

DMSO

Storage

at -20°C

Customer Product Validations & Biological Datas

Source

ACS Chem Biol (2016). Figure 5. Neratinib (Abmole Bioscience)

Method

FECH inhibition assay

Cell Lines

K562 Cells

Concentrations

1 μM

Incubation Time

6 days

Results

In line with the Kd values obtained from kinobead experiments, Vemurafenib, CUDC 101, Cyc-116, Linsitinib, MK-2461, Neratinib, GSK-690693, and Crenolanib decreased FECH binding to the PPIX beads by 50% or more compared to the DMSO control.

Rating

Source

ACS Chem Biol (2016). Figure 4. Neratinib (Abmole Bioscience)

Method

FECH inhibition assay

Cell Lines

K562 Cells

Concentrations

1 μM

Incubation Time

6 days

Results

We then subjected MK-2461, Neratinib, and Linsitinib to the same assay (1 μM for 6 days), and each drug clearly led to reduced heme levels indicating inhibition of FECH activity in K562 cells with Neratinib showing the most potent effect (60% reduction).

Rating

Source

ACS Chem Biol (2016). Figure 3. Neratinib (Abmole Bioscience)

Method

FECH binding

Cell Lines

K562 Cells

Concentrations

Incubation Time

Results

The basis of this assay is that proteins bound to a drug molecule are stabilized against heat induced unfolding and aggregation, and the extent of this protection is dose dependent.

Rating

Source

ACS Chem Biol (2016). Figure 2. Neratinib (Abmole Bioscience)

Method

FECH binding

Cell Lines

Concentrations

Incubation Time

Results

Figure 2b−f show the full dose response data for five selected inhibitors, and the complete set of these plots can be found in Supporting Information Figure S1 and Table S1.

Pertuzumab, a humanized monoclonal antibody and the first in the class of agents called the HER2 dimerization inhibitors, impairs the ability of HER2 to bind to other members of the HER family, MW: 148 KD.